Our long-term goal is to understand the process of DNA repair promoted by the system of homologous recombination in human cells. The system of homologous recombination is responsible for the repair of DNA double-stranded breaks (DSB) and inter-strand cross-links (ICL), the most harmful DNA lesions. RAD54 is one of the key proteins of homologous recombination. RAD54 knock-outs significantly increase mice sensitivity to ionizing radiation and ICL-inducing agents. While the biochemical activities of RAD54 are well characterized, its specific cellular functions remain to be elucidated. The goal of this proposal is to develop specific small- molecule inhibitors of human RAD54 protein using high throughput screening (HTS) of chemical libraries. Specific inhibitors present a valuable tool to study RAD54 functions in human cells. Since DSB- and ICL- inducing agents are commonly used in anticancer therapy, specific RAD54 inhibitors may also help to increase the therapy efficacy. We previously showed that RAD54 promotes branch migration of Holliday junctions, key homologous recombination intermediates, using the energy of ATP hydrolysis. Branch migration of Holliday junctions constitutes an important step that is required for completion of homologous recombination events. In order to identify inhibitors of the RAD54 branch migration activity by HTS we developed an in vitro FRET-based primary assay. The assay was validated in the pilot screen of the MLPCN compound library (Z' >0.8) that yielded ten tentative RAD54 inhibitors (hits). Robust secondary and tertiary assays have been developed to evaluate the biological significance of hits. To eliminate false positives due to fluorescence interference, a secondary assay using DNA substrates with a different fluorophore than that in the primary assay will be used. Additionally, an orthogonal assay using radioactively-labeled Holliday junction substrates and gel-electrophoresis will help to prioritize true hits. We have demonstrated the efficiency of both assays in elimination of false positives. The specificity of th selected inhibitors will be examined using human RAD51 protein that is structurally unrelated to RAD54. Cell-based systems will be used to determine the effect of confirmed RAD54 inhibitors on DNA repair, homologous recombination, and cell viability. The Structure Activity Relationships (SAR) of the prioritized inhibitors will be developed to increase their selectivity and potency. In continuation of this grant, the mechanisms of RAD54 inhibition by the selected compounds will be investigated using several tertiary assays including ATP hydrolysis, DNA binding, RAD54 oligomerization, and DNA translocation. The therapeutic potential of the prioritized compounds will be examined using immune-deficient mice with transplanted human xenografts.